Using diverse U.S. beef cattle genomes to identify missense mutations in EPAS1, a gene associated with pulmonary hypertension

Abstract

Discuss this article AbstractThe availability of whole genome sequence (WGS) data has made it possible to discover protein variants . However, existing bovine WGS databases do in silico not show data in a form conducive to protein variant analysis, and tend to under represent the breadth of genetic diversity in global beef cattle. Thus, our first aim was to use 96 beef sires, sharing minimal pedigree relationships, to create a searchable and publicly viewable set of mapped genomes relevant for 19 popular breeds of U.S. cattle. Our second aim was to identify protein variants encoded by the bovine endothelial PAS domain-containing protein 1 gene ( ), a gene associated with pulmonary hypertension in Angus cattle. The EPAS1 identity and quality of genomic sequences were verified by comparing WGS genotypes to those derived from other methods. The average read depth, genotype scoring rate, and genotype accuracy exceeded 14, 99%, and 99%, respectively. The 96 genomes were used to discover four amino acid variants encoded by (E270Q, P362L, A671G, and L701F) and confirm two EPAS1 variants previously associated with disease (A606T and G610S). The six missense mutations were verified with matrix-assisted laser EPAS1 desorption/ionization time-of-flight mass spectrometry assays, and their frequencies were estimated in a separate collection of 1154 U.S. cattle representing 46 breeds. A rooted phylogenetic tree of eight polypeptide sequences provided a framework for evaluating the likely order of mutations and potential impact of alleles on the adaptive response to chronic EPAS1 hypoxia in U.S. cattle. This public, whole genome resource facilitates in silico identification of protein variants in diverse types of U.S. beef cattle, and provides a means of translating WGS data into a practical biological and evolutionary context for generating and testing hypotheses.

A bovine CD18 signal peptide variant with increased binding activity to Mannheimia hemolytica leukotoxin

“…After sequencing, the raw reads were filtered to remove adaptor sequences, contaminating dimer sequences, and low-quality reads. The DNA sequence alignment process was similar to that previously reported 20 . FASTQ files were aggregated for each sample and DNA sequences were aligned individually to the bovine reference assembly UMD3.1 28 with the Burrows-Wheeler aligner (BWA) aln algorithm version 0.7.12 29 , then merged and collated with bwa sampe.…”

“…Two panels of DNAs were used to determine ITGB2 genotypes from U.S. cattle. The first was a previously described panel of 96 unrelated beef cattle from 19 popular U.S. beef breeds that had already been characterized by whole-genome sequencing 20 . This identified predicted coding changes throughout the ITGB2 gene.…”

“…Whole genome sequencing of BL3 cells (a bovine lymphoma cell line; kindly provided by Dr. Subramaniam Srikumaran) was accomplished with methods as described elsewhere 20 . Briefly, genomic DNA was used to make a 500 bp paired-end library and sequenced with a massively parallel sequencing machine and high-output kits (NextSeq500, two by 150 paired-end reads, Illumina, San Diego, CA, USA) until a minimum of 40 GB of data with greater than Q20 quality, was collected.…”

“…Viewing the aligned sequences and detecting variants was accomplished with the IGV software and a browser developed for this purpose. Briefly, public internet sites at the USDA, ARS, USMARC were used in combination with open source software installed on a laptop computer and recorded manually in a spreadsheet as previously described 20 . A Java Runtime Environment (Oracle Corporation, Redwood Shores, CA, USA) was first installed on the computer.…”

Chromosome-length haplotigs for yak and cattle from trio binning assembly of an F1 hybrid

“…For example if the sire was homozygous for allele A, the dam homozygous for allele B, and the progeny homozygous for B, in the absence of a genotyping error, this pattern suggests that the reported sire is not in fact the sire. We expect some genotyping errors [33,34] , but whatever exclusions are identified when analyzing the verifiable trio should be dwarfed in number when one of the actual parents is swapped in the analysis with an unrelated animal. For this comparison we did trio analysis of the yak × cattle offspring versus the reported Highland sire and yak dam as well as the reported dam versus four unrelated Highland bulls, and the reported sire versus an unrelated yak dam.…”